Type 1 diabetes is a chronic metabolic disease associated with development of severe complications (Nathan et al., 2009). It has been shown that type 1 diabetes can be cured by the transplantation of the pancreas or isolated islets of Langerhans. Nonetheless, the success of pancreas and islet transplantation is limited by the shortage of organ donors and the need for systemic immunosuppressive therapy (Robertson, 2010) and is therefore restricted to few patients (CITR, 2007).
If enough donor islets were available, type 2 diabetics also would profit from islet transplantation, if insulin supplementation is required, when other treatments are insufficient, or if they suffer from specific β-cell defects (Bottino & Trucco, 2005).
Limited availability of human donor organs may be overcome by the use of animals, in particular pigs, as organ donors. Pig-to-human xenotransplantation faces the problem of strong rejection predominantly by direct T cell recognition of pig major histocompatibility complex (MHC) and indirect T cell response to xenogeneic antigens presented by the recipient antigen-presenting cells (APCs) (Clarkson & Sayegh, 2005).
Recent advances in immunosuppressive therapies provided evidence that transplanted porcine islets can promote long-lasting cure of diabetes in nonhuman primates (Cardona et al., 2006, Hering et al., 2005, Cardona et al., 2007). However, the currently used intensive immunosuppressive regimen in pig islet transplantation may have severe side effects in humans and cannot be transferred into clinical practice. Blockade of the B7/CD28 co-stimulatory pathway by LEA29Y, a high affinity variant of the CTLA-4Ig fusion protein (Larsen et al., 2005), has been shown to be effective in clinical trials following kidney transplantation (Durrbach et al., 2010, Vincenti et al., 2010) and in porcine islet transplantation studies (Cardona et al., 2006 and 2007; Tchorch-Yutsis, 2009).
Phelps et al. (2009) disclose the production and characterization of transgenic pigs expressing porcine CTLA4-Ig. However, the transgenic pigs exhibited robust and ubiquitous expression of pCTLA4-Ig and the expression of pCTLA4-Ig resulted in acute susceptibility to opportunistic pathogens due at least in part to a significantly compromised humoral immune status. The authors found that, as CTLA4-Ig molecule is known to have immunosuppressive activity, high levels of pCTLA4-Ig expression in the blood, as well as defective development related to exposure to pCTLA4-Ig in utero, contributed to the reduced immune status. Therefore, prophylactic treatment with antibiotics appears to be necessary to promote survival of disease-free transgenic pigs to a size optimal for organ procurement for transplantation.
US 2009/0186097 A1 discloses the use of two vector constructs containing CTLA4-Ig, driven by the two different promoters, for transfecting fetal fibroblast cell lines. According to Example 5 of US 2009/0186097, cell populations and cloned colonies that screen positive for the presence of a CTLA4-Ig construct can be used as nuclear donors to produce CTLA4-Ig transgenic pigs by nuclear transfer. However, no transgenic pigs are described in the application. Only one kind of transgenic pigs which were generated using a plasmid construct for CTLA4-Ig driven by the chicken beta-actin promoter has later been published and turned out to be not a realistic option for xenotransplantation, see Phelps et al., 2009 as discussed above.
Thus, there is a need in the art for improved means and methods of treating diabetes in patients, in particular for providing suitable donor animals.